Requirement of Smad3 and CREB-1 in mediating transforming growth factor-β (TGFβ) induction of TGFβ3 secretion

Because increased transforming growth factor-beta (TGF beta) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGF beta production is important. Previous studies have identified the precise signaling components and promoter elements required for TGF beta induction of TGF beta 1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). To determine how regulation of TGF beta 3 expression differs from that of TGF beta 1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGF beta 3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGF beta 3 promoter was required for TGF beta-inducible TGF beta 3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGF beta-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGF beta 3 promoter (-100 to +1), whereas Smad3 bound at this site only after TGF beta stimulation. In addition, inhibition of JNK and p38 suppressed TGF beta induction of TGF beta 3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGF beta stimulation of TGF beta 3 promoter reporter activity and TGF beta 3 production. Our results indicate that TGF beta activation of the TGF beta 3 promoter CRE site, which leads to TGF beta 3 production, is required for TGF beta RII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.